During 2011, 96 sclerotial isolates of Rhizoctonia solani were collected from potato tubers from all main potato-cultivating regions of Cyprus. All isolates were found to be multinucleate. Characterization of anastomosis groups (AG) based on hyphal anastomosis reactions showed that 91 isolates belonged to AG3 and 5 to AG4. Sequence analysis of the internal transcribed spacer (ITS) regions (ITS1 and ITS2) of ribosomal DNA (rDNA) of 68 isolates confirmed the prevalence of AG3. In addition, phylogenetic analysis found that AG3 isolates were of the potato type, distinctly separated from the AG3 tobacco type, while AG4 isolates were separated into two different subgroups (HGI and HGII). Temperature studies showed that isolates belonging to both AG4 subgroups had significantly higher optimum growth temperatures compared with AG3. In vitro sensitivities to the fungicide pencycuron, in terms of concentrations where 50% growth inhibition was observed, ranged from 0.012 to 0.222 μg/ml. Pathogenicity and aggressiveness of the isolates was determined on 'Annabelle' potato sprouts and seedlings of a number of selected hosts, based on crop rotations followed in Cyprus. The majority of the isolates were pathogenic to potato sprouts, with disease severity (DS) values ranging from 0 to 88%. Mean DS values were statistically different among AG and subgroups, with AG4-HGI (69.25%) and AG4-HGII (3.12%) being the most and least aggressive, respectively. However, AG4-HGII isolates were the most aggressive in all rotational hosts tested, while AG3 isolates were the least aggressive. More specifically, the highest DS levels by AG4-HGI were recorded to barley, by AG4-HGII to lettuce and melon, and by AG3 isolates to vetch. This is the first comprehensive study to elucidate the AG composition, pathogenicity and other biological aspects of R. solani isolates associated with potato black scurf in Cyprus.